DNA sequencing in HydroLink matrices: extension of reading ability to greater than 600 nucleotides.
All the systems for optimizing DNA sequencing published so far have introduced modifications regarding: (i) linearization of band migration via ionic strength gradients or wedge-shaped gels; (ii) automatization of band reading via introduction of fluorescent probes; (iii) direct blotting analysis; (iv) pulsed electric fields and (v) discontinuous buffer systems. In all these systems, DNA sequence reading with an accuracy of ca. 98% rarely exceeds a length of 350 bases. We have chosen, in order to increase the reading ability of a single gel, to manipulate the characteristics of the gel matrix. The Seq-HydroLink gel formation here reported allows optimal reading, from a single gel run, of at least 600 bases. In order to guarantee this reading ability in a single run, the upper and lower ends of the ladder are time-resolved, i.e. the same sample is applied to the gel matrix at three different time intervals. The present system represents an increase of at least 30% in reading ability as compared with any type of polyacrylamide gel formulation so far reported.
Gelfi
C
C
Department Biomedical Sciences and Technologies, University of Milano.
Canali
A
A
Righetti
P C
PC
Vezzoni
P
P
Smith
C
C
Mellon
M
M
Jain
T
T
Shorr
R
R
D001435:Bacteriophages / Q000235:genetics; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D009710:Nucleotide Mapping
D003432:Cross-Linking Reagents; D004247:DNA